Integrative genomic analyses of European intrahepatic cholangiocarcinoma: Novel ROS1 fusion gene and PBX1 as prognostic marker.

BACKGROUND: Cholangiocarcinoma (CCA) is a fatal cancer of the bile duct with a poor prognosis owing to limited therapeutic options. The incidence of intrahepatic CCA (iCCA) is increasing worldwide, and its molecular basis is emerging. Environmental factors may contribute to regional differences in the mutation spectrum of European patients with iCCA, which are underrepresented in systematic genomic and transcriptomic studies of the disease. METHODS: We describe an integrated whole-exome sequencing and transcriptomic study of 37 iCCAs patients in Germany. RESULTS: We observed as most frequently mutated genes ARID1A (14%), IDH1, BAP1, TP53, KRAS, and ATM in 8% of patients. We identified FGFR2::BICC1 fusions in two tumours, and FGFR2::KCTD1 and TMEM106B::ROS1 as novel fusions with potential therapeutic implications in iCCA and confirmed oncogenic properties of TMEM106B::ROS1 in vitro. Using a data integration framework, we identified PBX1 as a novel central regulatory gene in iCCA. We performed extended screening by targeted sequencing of an additional 40 CCAs. In the joint analysis, IDH1 (13%), BAP1 (10%), TP53 (9%), KRAS (7%), ARID1A (7%), NF1 (5%), and ATM (5%) were the most frequently mutated genes, and we found PBX1 to show copy gain in 20% of the tumours. According to other studies, amplifications of PBX1 tend to occur in European iCCAs in contrast to liver fluke-associated Asian iCCAs. CONCLUSIONS: By analyzing an additional European cohort of iCCA patients, we found that PBX1 protein expression was a marker of poor prognosis. Overall, our findings provide insight into key molecular alterations in iCCA, reveal new targetable fusion genes, and suggest that PBX1 is a novel modulator of this disease.

ROS-induced translational regulation-through spatiotemporal differences in codon recognition-is a key driver of brown adipogenesis.

The role of translational regulation in brown adipogenesis is relatively unknown. Localized translation of mRNAs encoding mitochondrial components enables swift mitochondrial responses, but whether this occurs during brown adipogenesis, which involves massive mitochondrial biogenesis, has not been explored. Here, we used ribosome profiling and RNA-Seq, coupled with cellular fractionation, to obtain spatiotemporal insights into translational regulation. During brown adipogenesis, a translation bias towards G/C-ending codons is triggered first in the mitochondrial vicinity by reactive oxygen species (ROS), which later spreads to the rest of the cell. This translation bias is induced through ROS modulating the activity of the tRNA modification enzyme, ELP3. Intriguingly, functionally relevant mRNAs, including those encoding ROS scavengers, benefit from this bias; in so doing, ROS-induced translation bias both fuels differentiation and concurrently minimizes oxidative damage. These ROS-induced changes could enable sustained mitochondrial biogenesis during brown adipogenesis, and explain in part, the molecular basis for ROS hormesis.

Dengue virus exploits the host tRNA epitranscriptome to promote viral replication.

The 40-50 RNA modifications of the epitranscriptome regulate posttranscriptional gene expression. Here we show that flaviviruses hijack the host tRNA epitranscriptome to promote expression of pro-viral proteins, with tRNA-modifying ALKBH1 acting as a host restriction factor in dengue virus infection. Early in the infection of human Huh-7 cells, ALKBH1 and its tRNA products 5-formylcytidine (f5C) and 2'-O-methyl-5-formylcytidine (f5Cm) were reduced. ALKBH1 knockdown mimicked viral infection, but caused increased viral NS3 protein levels during infection, while ALKBH1 overexpression reduced NS3 levels and viral replication, and increased f5C and f5Cm. Viral NS5, but not host FTSJ1, increased f5Cm levels late in infection. Consistent with reports of impaired decoding of leucine UUA codon by f5Cm-modified tRNALeu(CAA), ALKBH1 knockdown induced translation of UUA-deficient transcripts, most having pro-viral functions. Our findings support a dynamic ALKBH1/f5Cm axis during dengue infection, with virally-induced remodeling of the proteome by tRNA reprogramming and codon-biased translation.

An RNA modification enzyme directly senses reactive oxygen species for translational regulation in Enterococcus faecalis.

Bacteria possess elaborate systems to manage reactive oxygen and nitrogen species (ROS) arising from exposure to the mammalian immune system and environmental stresses. Here we report the discovery of an ROS-sensing RNA-modifying enzyme that regulates translation of stress-response proteins in the gut commensal and opportunistic pathogen Enterococcus faecalis. We analyze the tRNA epitranscriptome of E. faecalis in response to reactive oxygen species (ROS) or sublethal doses of ROS-inducing antibiotics and identify large decreases in N2-methyladenosine (m2A) in both 23 S ribosomal RNA and transfer RNA. This we determine to be due to ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Genetic knockout of RlmN gives rise to a proteome that mimics the oxidative stress response, with an increase in levels of superoxide dismutase and decrease in virulence proteins. While tRNA modifications were established to be dynamic for fine-tuning translation, here we report the discovery of a dynamically regulated, environmentally responsive rRNA modification. These studies lead to a model in which RlmN serves as a redox-sensitive molecular switch, directly relaying oxidative stress to modulating translation through the rRNA and the tRNA epitranscriptome, adding a different paradigm in which RNA modifications can directly regulate the proteome.

Peer mentoring programs for nursing students: A mixed methods systematic review.

BACKGROUND: Peer mentoring in nursing is imperative to both mentors' and mentees' personal and professional development. Yet, there is a dearth of reviews appraising the relevant qualitative and quantitative studies reported in the literature. OBJECTIVES: To synthesize the best evidence exploring the impacts of peer mentoring programs on nursing students. DESIGN: A mixed-method systematic review. DATA SOURCES: Published and unpublished literature written in English between January 2011 and May 2022 were identified from nine databases, including PubMed, CINAHL, Cochrane Library, Embase, and PsycINFO. REVIEW METHODS: A systematic search strategy was applied in June 2021. Two reviewers independently screened and selected the eligible studies focusing on nursing students in higher education institutions who participated in peer mentoring programs. We included studies of quantitative, qualitative, or mixed methods and explored the main outcomes of peer mentoring programs. Eligible studies were appraised independently using the Joanna Briggs Institute (JBI) Critical Appraisal Checklists. Two independent reviewers extracted relevant data using a standardized form. A meta-analysis, narrative synthesis, and meta-aggregation were then conducted, followed by a convergent segregated approach to integrate the findings. RESULTS: Thirty-one studies were selected for analysis. We aggregated four sets of synthesized findings from fourteen categories. A meta-analysis of the data showed that the intervention group experienced significant improvement in stress levels. In addition, the integrated results revealed peer mentors, with the support of academia, served as rich resources and support for peer mentees. CONCLUSION: This review highlights the importance of providing support to peer mentors to help them achieve the desired outcomes of peer mentoring while they cater to the needs of nursing student mentees.

A warm-start digital CRISPR/Cas-based method for the quantitative detection of nucleic acids.

Nucleic acids-based molecular diagnostic tools incorporating the CRISPR/Cas system are being developed as rapid and sensitive methods for pathogen detection. However, most CRISPR/Cas-based diagnostics lack quantitative detection ability. Here, we report Warm-Start RApid DIgital Crispr Approach (WS-RADICA) for the rapid, sensitive, and quantitative detection of nucleic acids. WS-RADICA detected as little as 1 copy/µl SARS-CoV-2 RNA in 40 min (qualitative detection) or 60 min (quantitative detection). WS-RADICA can be easily adapted to various digital devices: two digital chips were evaluated for both DNA and RNA quantification, with linear dynamic ranges of 0.8-12777 copies/µL for DNA and 1.2-18391 copies/µL for RNA (both R2 values > 0.99). Moreover, WS-RADICA had lower detection limit and higher inhibitor tolerance than a bulk RT-LAMP-Cas12b reaction and similar performance to RT-qPCR and RT-dPCR. To prove its performance on nucleic acids derived from live virus, WS-RADICA was also validated to detect and quantify human adenovirus and herpes simplex virus. Given its speed, sensitivity, quantification capability, and inhibitor tolerance, WS-RADICA shows great promise for a variety of applications requiring nucleic acid quantification.

Association between family history and lung cancer risk among Chinese women in Singapore.

Risk factors of lung cancer unrelated to smoking are not well-studied, especially among women. Family history has been shown to play a role in predisposing individuals to lung cancer, but this relationship has not been investigated in the Southeast Asian population. A total of 1159 women were recruited in a case-control study conducted in public hospitals in Singapore from 2005 to 2008. After excluding participants with incomplete family history information, 374 cases and 785 controls remained in the final analysis. Adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were calculated using logistic regression, adjusting for potential confounders. Overall, family history of lung cancer was associated with a higher risk for lung cancer (aOR 2.08, 95% CI 1.25-3.47). When stratified by smoking status, a significant association was observed among never-smokers (aOR 2.78, 95% CI 1.57-4.90). Further stratification by fruit consumption identified a significant association between family history of lung cancer and higher risk of lung cancer among never-smokers who had low fruit consumption (aOR 3.09, 95% CI 1.37-7.01). Our findings suggest that family history of lung cancer is a significant risk factor for lung cancer in Singaporean Chinese women, especially among never-smokers.

Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids.

Rapid diagnostics of adventitious agents in biopharmaceutical/cell manufacturing release testing and the fight against viral infection have become critical. Quantitative real-time PCR and CRISPR-based methods rapidly detect DNA/RNA in 1 h but suffer from inter-site variability. Absolute quantification of DNA/RNA by methods such as digital PCR reduce this variability but are currently too slow for wider application. Here, we report a RApid DIgital Crispr Approach (RADICA) for absolute quantification of nucleic acids in 40-60 min. Using SARS-CoV-2 as a proof-of-concept target, RADICA allows for absolute quantification with a linear dynamic range of 0.6-2027 copies/µL (R2 value > 0.99), high accuracy and low variability, no cross-reactivity to similar targets, and high tolerance to human background DNA. RADICA's versatility is validated against other targets such as Epstein-Barr virus (EBV) from human B cells and patients' serum. RADICA can accurately detect and absolutely quantify EBV DNA with similar dynamic range of 0.5-2100 copies/µL (R2 value > 0.98) in 1 h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based detection. RADICA therefore enables rapid and sensitive absolute quantification of nucleic acids which can be widely applied across clinical, research, and biomanufacturing areas.

Creating a toolkit with stakeholders for leveraging tablet computers to support person-centred dementia care in hospitals.

INTRODUCTION: People with dementia may refuse care because they feel overwhelmed by an unfamiliar environment. Everyday technology such as tablets have the potential to support person-centred dementia care in hospitals. AIMS: We aimed to identify barriers and enabling factors in order to develop a toolkit to support the use of tablets in engaging individual and group activities, especially to play family videos, for hospitalized older people with dementia. METHODS: A participatory action research approach was employed. We facilitated staff focus groups and conducted interviews with stakeholders. A toolkit was developed based on participants' perspectives on how to support successful adoption. RESULTS: Our analysis identified two enabling factors: users' engagement in developing a toolkit for support and adapting implementation to meet local needs. Barriers included staff and family inexperience, mechanical instability of hardware, issues around privacy and data access, technology use and personalization of messages. The toolkit includes short videos, a brochure for family caregivers, and a pocket card for staff.Discussion and implications: Staff, family and patients start with varying levels of experience with the use of tablets, making education and support vitally important to implementation. Health organizations should involve staff, patients, and families to find practical solutions.

Association between family history of lung cancer and lung cancer risk: a systematic review and meta-analysis.

BACKGROUND: Familial risk of lung cancer has been widely studied but the effects of sociodemographic factors and geographical regions are largely unknown. METHODS: PubMed and Embase were systematically searched until 1st October 2019. A total of 84 articles were identified and (19 cohort and 66 case control studies) included in this systematic review and meta-analysis. Pooled summary estimates and 95% confidence intervals were estimated, and the analysis was stratified by sociodemographic factors and geographical regions. RESULTS: Geographical regions, sex, age of proband, smoking status, type of first-degree relatives, number of affected relatives, and early onset of lung cancer in affected relatives were significant determinants of familial risk of lung cancer. Higher risk of familial lung cancer was found among Asians as compared to non-Asians, younger individuals (age≤50) as compared with older individuals (age>50), individuals with ≥2 affected relatives as compared with individuals with one affected relative, ever-smokers as compared with never-smokers, Asian females as compared with Western females, and never-smokers in Asia as compared with never-smokers in the West. CONCLUSIONS: Familial risk of lung cancer is influenced by both genetic and environmental factors. Future studies should control for environmental factors such as air pollution and environmental tobacco smoke which are prevalent in Asia.

Governing offshore fish aggregating devices in the Eastern Caribbean: Exploring trade-offs using a qualitative network model.

The use of moored Fish Aggregating Devices (FADs) in small-scale fisheries is a potential solution to food security concerns, economic development needs, and the overexploitation of nearshore coastal fisheries in the Eastern Caribbean. However, moored FADs also generate novel and largely unstudied governance challenges involving (1) the provisioning of FADs, (2) fisheries resource appropriation, (3) human wellbeing, and (4) food web impacts. We examine the relative performance of three governance scenarios to address these challenges: private-individual, community-based, and top-down governance. We construct a qualitative network model (QNM) of the fishery based on semi-structured interviews (n = 60) with fishers and fisheries managers, established food web and economic models, and expert knowledge. We simulate the social-ecological impacts of the three governance scenarios. The models suggest that community-based and top-down governance scenarios result in low levels of conflict, but provide limited incentives to develop and maintain moored FADs. The private-individual governance scenario tends to increase conflict and incentives for monitoring FADs, but has no impact on incentives for maintaining and deploying FADs.

An integrative model of pathway convergence in genetically heterogeneous blast crisis chronic myeloid leukemia.

Targeted therapies against the BCR-ABL1 kinase have revolutionized treatment of chronic phase (CP) chronic myeloid leukemia (CML). In contrast, management of blast crisis (BC) CML remains challenging because BC cells acquire complex molecular alterations that confer stemness features to progenitor populations and resistance to BCR-ABL1 tyrosine kinase inhibitors. Comprehensive models of BC transformation have proved elusive because of the rarity and genetic heterogeneity of BC, but are important for developing biomarkers predicting BC progression and effective therapies. To better understand BC, we performed an integrated multiomics analysis of 74 CP and BC samples using whole-genome and exome sequencing, transcriptome and methylome profiling, and chromatin immunoprecipitation followed by high-throughput sequencing. Employing pathway-based analysis, we found the BC genome was significantly enriched for mutations affecting components of the polycomb repressive complex (PRC) pathway. While transcriptomically, BC progenitors were enriched and depleted for PRC1- and PRC2-related gene sets respectively. By integrating our data sets, we determined that BC progenitors undergo PRC-driven epigenetic reprogramming toward a convergent transcriptomic state. Specifically, PRC2 directs BC DNA hypermethylation, which in turn silences key genes involved in myeloid differentiation and tumor suppressor function via so-called epigenetic switching, whereas PRC1 represses an overlapping and distinct set of genes, including novel BC tumor suppressors. On the basis of these observations, we developed an integrated model of BC that facilitated the identification of combinatorial therapies capable of reversing BC reprogramming (decitabine+PRC1 inhibitors), novel PRC-silenced tumor suppressor genes (NR4A2), and gene expression signatures predictive of disease progression and drug resistance in CP.

Robust CTCF-Based Chromatin Architecture Underpins Epigenetic Changes in the Heart Failure Stress-Gene Response.

BACKGROUND: The human genome folds in 3 dimensions to form thousands of chromatin loops inside the nucleus, encasing genes and cis-regulatory elements for accurate gene expression control. Physical tethers of loops are anchored by the DNA-binding protein CTCF and the cohesin ring complex. Because heart failure is characterized by hallmark gene expression changes, it was recently reported that substantial CTCF-related chromatin reorganization underpins the myocardial stress-gene response, paralleled by chromatin domain boundary changes observed in CTCF knockout. METHODS: We undertook an independent and orthogonal analysis of chromatin organization with mouse pressure-overload model of myocardial stress (transverse aortic constriction) and cardiomyocyte-specific knockout of Ctcf. We also downloaded published data sets of similar cardiac mouse models and subjected them to independent reanalysis. RESULTS: We found that the cardiomyocyte chromatin architecture remains broadly stable in transverse aortic constriction hearts, whereas Ctcf knockout resulted in ≈99% abolition of global chromatin loops. Disease gene expression changes correlated instead with differential histone H3K27-acetylation enrichment at their respective proximal and distal interacting genomic enhancers confined within these static chromatin structures. Moreover, coregulated genes were mapped out as interconnected gene sets on the basis of their multigene 3D interactions. CONCLUSIONS: This work reveals a more stable genome-wide chromatin framework than previously described. Myocardial stress-gene transcription responds instead through H3K27-acetylation enhancer enrichment dynamics and gene networks of coregulation. Robust and intact CTCF looping is required for the induction of a rapid and accurate stress response.

Lifestyle modifications: coordinating the tRNA epitranscriptome with codon bias to adapt translation during stress responses.

Cells adapt to stress by altering gene expression at multiple levels. Here, we propose a new mechanism regulating stress-dependent gene expression at the level of translation, with coordinated interplay between the tRNA epitranscriptome and biased codon usage in families of stress-response genes. In this model, auxiliary genetic information contained in synonymous codon usage enables regulation of codon-biased and functionally related transcripts by dynamic changes in the tRNA epitranscriptome. This model partly explains the association between synchronous stress-dependent epitranscriptomic marks and significant multi-codon usage skewing in families of translationally regulated transcripts. The model also predicts translational adaptation during viral infections.

Elucidating the genomic architecture of Asian EGFR-mutant lung adenocarcinoma through multi-region exome sequencing.

EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates.

The effect of badminton-specific exercise on badminton short-serve performance in competition and practice climates.

This study examined the effects of changes in physiological and psychological arousal on badminton short-serve performance in competitive and practice climates. Twenty competitive badminton players (10 males and 10 females) volunteered to participate in the study following ethics approval. After familiarisation, badminton short-serve performance was measured at rest, mid-way through and at the end of a badminton-specific exercise protocol in two conditions; competition vs. practice. Ratings of cognitive and somatic anxiety were assessed at three time points prior to badminton short-serve performance using the Mental Readiness Form 3. Heart rate and rating of perceived exertion (RPE) were assessed during the exercise protocol. Results indicated that better short-serve performance was evident in practice compared to competition (P = .034). RPE values were significantly higher in the competition condition compared to practice (P = .007). Cognitive anxiety intensity was significantly lower post-exercise in the practice condition compared to competition (P = .001). Cognitive anxiety direction showed greater debilitation post-exercise in the competition condition compared to practice (P = .01). Somatic anxiety intensity increased from pre-, to mid- to post-exercise (P = .001) irrespective of condition. This study suggests that badminton serve performance is negatively affected when physiological arousal, via badminton-specific exercise, and cognitive anxiety, via perceived competition, are high.

Loss of Bladder Epithelium Induced by Cytolytic Mast Cell Granules.

Programmed death and shedding of epithelial cells is a powerful defense mechanism to reduce bacterial burden during infection but this activity cannot be indiscriminate because of the critical barrier function of the epithelium. We report that during cystitis, shedding of infected bladder epithelial cells (BECs) was preceded by the recruitment of mast cells (MCs) directly underneath the superficial epithelium where they docked and extruded their granules. MCs were responding to interleukin-1ß (IL-1ß) secreted by BECs after inflammasome and caspase-1 signaling. Upon uptake of granule-associated chymase (mouse MC protease 4 [mMCPT4]), BECs underwent caspase-1-associated cytolysis and exfoliation. Thus, infected epithelial cells require a specific cue for cytolysis from recruited sentinel inflammatory cells before shedding.

Global re-wiring of p53 transcription regulation by the hepatitis B virus X protein.

BACKGROUND: The tumour suppressor p53 is a central player in transcription regulation and cell fate determination. By interacting with p53 and altering its sequence-specific binding to the response elements, the hepatitis B virus X protein (HBx) was reported to re-direct p53 regulation of some genes. RESULTS: Coupling massively parallel deep sequencing with p53 chromatin immunoprecipitation, we demonstrate that HBx modulates global p53 site selection and that this was strongly influenced by altered interaction with transcription co-factors/co-regulators as well as post-translational modifications. Specifically, HBx attenuated p53-TBP-RB1 transcription complex recruitment and interaction and this was associated with hyper-phosphorylation of p53 at serine 315 by HBx. Concurrently, HBx enhanced p53 DNA occupancy to other response elements either alone by displacing specific transcription factors such as CEBPB and NFkB1, or in complex with distinct interacting co-factors Sp1, JUN and E2F1. Importantly, re-wiring of p53 transcription regulation by HBx was linked to the deregulation of genes involved in cell proliferation and death, suggesting a role of HBx in errant cell fate determination mediated by altered p53 site selection of target genes. CONCLUSIONS: Our study thus presents first evidence of global modes of p53 transcription alteration by HBx and provides new insights to understand and potentially curtail the viral oncoprotein.

Post-translational control of transcription factors: methylation ranks highly.

Methylation of lysine and arginine residues on histones has long been known to determine both chromatin structure and gene expression. In recent years, the methylation of non-histone proteins has emerged as a prevalent modification which impacts on diverse processes such as cell cycle control, DNA repair, senescence, differentiation, apoptosis and tumourigenesis. Many of these non-histone targets represent transcription factors, cell signalling molecules and tumour suppressor proteins. Evidence now suggests that the dysregulation of methyltransferases, demethylases and reader proteins is involved in the development of many diseases, including cancer, and several of these proteins represent potential therapeutic targets for small molecule compounds, fuelling a recent surge in chemical inhibitor design. Such molecules will greatly help us to understand the role of methylation in both health and disease.

High-depth sequencing of over 750 genes supports linear progression of primary tumors and metastases in most patients with liver-limited metastatic colorectal cancer.

BACKGROUND: Colorectal cancer with metastases limited to the liver (liver-limited mCRC) is a distinct clinical subset characterized by possible cure with surgery. We performed high-depth sequencing of over 750 cancer-associated genes and copy number profiling in matched primary, metastasis and normal tissues to characterize genomic progression in 18 patients with liver-limited mCRC. RESULTS: High depth Illumina sequencing and use of three different variant callers enable comprehensive and accurate identification of somatic variants down to 2.5% variant allele frequency. We identify a median of 11 somatic single nucleotide variants (SNVs) per tumor. Across patients, a median of 79.3% of somatic SNVs present in the primary are present in the metastasis and 81.7% of all alterations present in the metastasis are present in the primary. Private alterations are found at lower allele frequencies; a different mutational signature characterized shared and private variants, suggesting distinct mutational processes. Using B-allele frequencies of heterozygous germline SNPs and copy number profiling, we find that broad regions of allelic imbalance and focal copy number changes, respectively, are generally shared between the primary tumor and metastasis. CONCLUSIONS: Our analyses point to high genomic concordance of primary tumor and metastasis, with a thick common trunk and smaller genomic branches in general support of the linear progression model in most patients with liver-limited mCRC. More extensive studies are warranted to further characterize genomic progression in this important clinical population.